This proposal is based on the following hypothesis: pluripotent cells are distinguished from their somatic cell counterparts by unique epigenotypes. Our hypothesis is based upon the following observations: First, ES cell histones have posttranslational modification (PTM) patterns that differ from somatic cells1. Globally increased acetylation levels suggest many genes within ES cells are in a "primed" state; further, developmental regulatory genes within ES cells have been identified with H3 lysine 27 trimethylation, a repressive epigenotype that is removed upon differentiation2. Second, distinct regions of chromosomes are characterized by epigenetic signatures. Centromeres, for example, have highly methylated histone tails and utilize an H3 variant to direct kinetochore formation3. Telomere length in mammalian cells is regulated by the activity of histone methyltransferases4, whereas human ES cell telomeres are immune to telomere shortening, regardless of the age of the cells in culture. Modern antibody-based techniques have allowed scientists to identify the presence or absence of a variety of histone modifications associated with a specific gene. However, these techniques cannot facilitate the identification of the PTM patterns across the intact histone tail (~1-50 residues) - imagine the code as a complete sentence, antibodies can identify letters or sometimes words, but these words and letters lack context. Recent developments in mass spectrometry-based technology, co-invented by the PI, have created a new tool to probe intact histone tails (1-50 residues) with unprecedented molecular detail and sensitivity (i.e., allows direct reading of the entire sentence). Here we combine this cutting-edge MS-based approach with large-scale human ES cell culture capacity, recently developed by Program PI Thomson research group, to determine the epigenotype(s) responsible for human ES cell pluripotency.